Theses and Dissertations from UMD
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Item SEASONAL NITROGEN CYCLING AND HOMEOSTASIS IN POPULUS: SOURCE-SINK COMMUNICATION(2018) Li, Gen; Coleman, Gary D; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Nitrogen (N) is an essential nutrient for plant growth, development and reproduction. Seasonal N cycling is an adaption to nutrient limitation and a feature of the perennial lifestyle of trees. Poplar (Populus) is a model system used to study forest tree genetics and molecular biology, including seasonal N cycling. The accumulation of Bark storage proteins (BSP) is a central feature of seasonal N cycling in poplar, yet our understanding of the contribution of the BSP storage pool to N remobilization during growth and mechanisms that regulate BSP accumulation, catabolism and N remobilization is limited. The research presented in this dissertation is directed towards advancing knowledge of the regulation of seasonal N cycling in poplar using a combination of experimental approaches. The role of the N storage pool to N remobilization was examined through N sink manipulations and the specific role of BSP storage was investigated by N source reduction via RNAi mediated knockdown of BSP accumulation. From this it was found that N remobilization from BSP significantly contributes to shoot growth following dormancy and initial shoot growth is source limited. Poplar bark transcriptome analyses during regrowth following dormancy revealed an enrichment for up-regulated genes associated with auxin transport and signaling. Based on the transcriptome analysis experiments that manipulate auxin production or polar auxin transport were performed and the results indicate that BSP catabolism and N remobilization likely involves polar auxin transport from expanding buds and shoots and auxin-mediated regulation of protease gene expression. Analysis of DNA microarrays of bark RNA during short-day (SD) induction of BSP gene expression was used to identify putative regulatory factors that may play a role of BSP accumulation. The transcriptome analyses indicated that SD represses the expression of genes involved in ethylene production as well as a reduction in bark ethylene biosynthesis. Additionally, treatment of excised stems with ACC or ethephon repressed BSP gene expression while AVG induced BSP gene expression. This repression was reduced in ethylene-insensitive poplars expressing Arabidopsis dominant gain-of-function allele etr1-1. Furthermore, transient expression of ERF12 and ERF41, two transcription factors with the greatest induction in SD treatment, in transgenic tobacco stably transformed with BSPA promoter fused with GUS resulted in enhanced GUS activity suggesting ERF12 and ERF41 may act as positive regulators of BSP gene expression. Since glutamine plays a pivotal role in N partitioning during N storage and remobilization, the possible role of the PII glutamine sensor was studied and it was found that the transcript levels of PII increased in bark during SD-induced leaf senescence and BSP accumulation. PII knockdown poplars using RNAi showed reduced glutamine-induced BSP gene expression. Moreover, glutamine-induced BSP gene expression was also inhibited by 2-OG, a PII-NAGK interaction antagonist, suggesting a possible role of PII-glutamine sensing in BSP accumulation. Taken together, this study provides important insights into the mechanism of seasonal N accumulation and remobilization in poplar.Item Investigating the molecular mechanism of RTE1 activation of the ethylene receptor ETR1 in Arabidopsis(2011) Chang, Jianhong; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The plant hormone ethylene plays a vital role in regulating plant growth and development as well as plant defense to biotic and abiotic stresses during the entire life of the plant. In Arabidopsis, ethylene is perceived by a family of five receptors, one of which is ETR1. The Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) gene is a positive regulator of ETR1. RTE1 encodes a novel integral membrane protein that interacts with ETR1 at the Golgi apparatus and the endoplasmic reticulum (ER). Genetic evidence indicates that RTE1 is required for the formation of a functional ETR1 receptor, whereas the other ethylene receptors in Arabidopsis do not require RTE1. But the molecular mechanism by which RTE1 specifically activates ETR1 remains unknown. I took different approaches to gain insights into the molecular function of RTE1 and the basis for the specificity for activating ETR1. In a library screen for RTE1–interacting proteins using the yeast split–ubiquitin assay, an ER–localized cytochrome b5 isoform (AtCb5–D) was identified. Cb5 is a small hemoprotein that functions in oxidation/reduction reactions. Mutants of three AtCb5 isoforms show phenotypes in ethylene responses that are similar to those of the rte1 mutant, suggesting the functional parallel between AtCb5 and RTE1 in ethylene signaling. Additional genetic analyses suggest that AtCb5 might act in the same pathway as RTE1 and that AtCb5 is specific to ETR1 like RTE1. Moreover, using a hemin–agarose affinity chromatography assay, I found that RTE1 homologs are able to bind heme in vitro, raising the possibility that RTE1 carries out redox with cytochrome b5s. I also found that the specificity for regulating ETR1 by RTE1 is largely due to a unique proline (P9) conserved only in ETR1 orthologs; introduction of P9 into the Arabidopsis ERS1 ethylene receptor was sufficient to convert ERS1 into an RTE1–dependent receptor. I propose that P9 may interfere with the proper folding of ETR1 EBD and formation of the ETR1 homodimer by affecting the conserved disulfide bond–forming cysteines (C4, C6) in the ETR1 homodimer. Taken together, our results suggest a model in which RTE1, together with cytochrome b5, promotes the active conformation of ETR1 through oxidative folding.Item Investigation of Ethylene Signal Transduction Mechanisms: Characterizing the Novel Gene AWE1 and Testing Hypothesis of Raf-like CTR1's Function In Vivo(2009) Kendrick, Mandy Danielle; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is a gaseous plant hormone affecting multiple plant processes. Sixteen years ago the first components of the ethylene signaling pathway, the receptor ETR1 and Raf-like kinase CTR1, were identified. Since then many additional components of the pathway have been elucidated through genetic screens. Recent discoveries suggest ethylene signaling, once thought to be a linear pathway from ethylene perception at the endoplasmic reticulum to transcriptional activation at the nucleus, is more complex with multiple auto-feedback loops and potential parallel kinase cascades downstream of the receptors. Although the genetic backbone of the pathway is well established, the signaling mechanisms of the components remain unclear. ETR1 displays histidine kinase activity in vitro and physically interacts with the next-known downstream component of the pathway, CTR1. However the histidine kinase activity of ETR1 is mostly dispensable for signaling to CTR1. How then is CTR1 activated? I proposed that additional proteins, like AWE1, play a role in ETR1 to CTR1 signaling, and that the non-catalytic, amino-terminal region of CTR1 is required both for activation through direct interaction with the ETR1 receptor complex and for auto-inhibition of CTR1 kinase activity. ASSOCIATES-WITH-ETR1 (AWE1) was isolated in a yeast-two-hybrid screen for ETR1-interacting proteins and was of specific interest because the AWE1 clone also interacted with a portion of CTR1. Protein-protein interaction studies and genetic analysis of an awe1 mutant support a role of AWE1 in repressing ethylene responses. However double mutant analysis, over-expression analysis, and protein sub-cellular localization studies suggest that AWE1's function in hypocotyl elongation and cell expansion is more general. AWE1's function may require ETR1 for proper regulation but is likely to lie outside of the direct step from ETR1 to CTR1. To investigate a role of the CTR1 amino-terminal region in CTR1 regulation, I constructed transgenes consisting of truncated ETR1 receptors fused to truncated or full length CTR1 and examined how those transgenes carrying the truncated CTR1 (kinase domain only) affected Arabidopsis seedling growth compared to those transgenes expressing full length CTR1. I concluded that the CTR1 amino-terminal region may have a role in autoregulation, but additional components are required for regulation of CTR1 signaling.Item Insights into the regulation of ethylene receptor signaling by RTE1(2008-10-10) rivarola, maximo lisandro; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is an important regulator of plant growth, development and responses to environmental stresses. The higher plant Arabidopsis thaliana perceives ethylene through five homologous receptors, which negatively regulate ethylene responses. The molecular mechanism by which these receptors signal to their next downstream component remains elusive. Genetic analyses have shown that the RTE1 locus is a positive regulator of ETR1. RTE1 encodes a novel protein of unknown molecular function, and is conserved in plants, animals and some protists. The goal of this research was to analyze the mechanisms involved in the regulation of ethylene receptor signaling by RTE1 and to enhance our understanding of the conserved cellular role of RTE1. Here we tested hypotheses for how RTE1 affects ETR1 and is specific to only ETR1, not the other ethylene receptor isoforms. We show that ETR1 and RTE1 gene expression patterns partially overlap and that the ETR1 receptor co-localizes with RTE1 within the cell. Moreover, RTE1 has no effect on ETR1 protein abundance or subcellular localization suggesting other mechanisms to regulate ETR1. We provide supporting evidence that RTE1 affects ETR1 signaling by restoring signaling of a non-functional ETR1 in an rte1 null through changes in ETR1 conformation(s). We next addressed the question of RTE1 specificity to ETR1. We discovered that ETR1 is surprisingly distinct from the other four ethylene receptor genes; in that RTE1-dependent mutations only confer insensitivity in ETR1 and not in the other ethylene receptors when the same mutations are introduced. In contrast, the RTE1-independent ETR1 insensitive mutations do give insensitivity in the closest receptor to ETR1, ERS1. Furthermore, we uncover that the ethylene binding domains are not completely interchangeable between ETR1 and ERS1. Our data point to a model in which RTE1 specifically promotes ETR1 signaling via conformational changes in a unique way that does not occur in other ethylene receptors. These findings highlight the importance and uniqueness of ETR1 signaling conformation(s) with respect to the other ethylene receptors, as well as advance our knowledge of RTE1 at the molecular and cellular level.Item REVERSION-TO-ETHYLENE-SENSITIVITY1: A Novel Regulator of Ethylene Receptor Function in Arabidopsis thaliana(2006-11-25) Resnick, Josephine; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is a plant hormone that has profound effects on plant growth and development. Genetic analysis has been central in the elucidation of the ethylene-signaling pathway, made possible through the isolation of ethylene-response mutants in Arabidopsis. This thesis focuses on elucidating the function of the Arabidopsis REVERSION-TO-ETHYLENE-SENSITIVITY1 (RTE1) locus, which was identified in a genetic screen for suppressors of the ethylene-insensitive receptor mutant etr1-2. The RTE1 gene was cloned by positional cloning and found to encode a novel integral membrane protein with homologs in plants and animals, but with no known molecular function. Our studies show that RTE1 is a negative regulator of the ethylene-response pathway, specifically acting as a positive regulator of the ETR1 ethylene receptor. Loss-of-function mutations in the RTE1 gene suppress the etr1-2 ethylene-insensitive phenotype, and genetic analysis suggests that loss of RTE1 results in a largely non-functional ETR1-2 mutant receptor. Similarly, wild-type ETR1 function appears to be greatly reduced in the absence of RTE1. Overexpression of the RTE1 gene confers weak ethylene insensitivity that is largely dependent on ETR1. rte1 mutations do not appear to affect the other four ethylene receptors of Arabidopsis, indicating that RTE1 specifically regulates ETR1. Sequence analysis revealed regions of conserved cysteine and histidine residues, and one rte1 loss-of-function mutant contains a point mutation at Cys161. Since such residues are common in metal binding proteins, we explored the possibility that RTE1 may be involved in facilitating the binding of an essential copper cofactor to the ETR1 receptor. However, experimental evidence suggests that this is not the likely role of RTE1. Interestingly, rte1 was unable to suppress the ethylene insensitive mutant etr1-1, indicating that the differences between etr1-2 and etr1-1 may hold a clue as to how RTE1 regulates ETR1. A suppression analysis of eleven additional etr1 insensitive mutants suggests that RTE1 plays a role in regulating signaling by the transmitter domain of ETR1. A possible role for RTE homologs in non-plant systems is also discussed, although more work is required to elucidate a detailed biochemical model for RTE1 action.